Unraveling tissue regeneration pathways using chemical genetics
Unraveling tissue regeneration pathways using chemical genetics
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DOI：
10.1074/jbc.m706640200
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发表时间：
2007-11-30
影响因子：
4.8
通讯作者：
Tanguay, Robert L.
中科院分区：
文献类型：
--
作者：
Mathew, Lijoy K.;Sengupta, Sumitra;Tanguay, Robert L.
确定再生所需的分子途径仍然是再生医学的巨大挑战之一。虽然基因突变对于识别一些分子途径是有用的，但再生途径的小分子探针可能提供一些优势，包括通过精确的时间控制来破坏途径功能的能力。然而，目前还没有一种适合快速通量小分子筛选的脊椎动物再生模型。我们在这里报告了斑马鱼早期生命阶段鳍再生模型的发展及其在筛选调节组织再生的小分子中的应用。通过筛选2000个生物活性小分子，我们确定了17个特异性抑制再生。这些化合物包括一簇糖皮质激素，我们证明糖皮质激素受体的短暂激活足以阻止再生，但只有当激活发生在伤口愈合/囊胚形成期间。此外，糖皮质激素受体的敲低恢复了非再生的、暴露于糖皮质激素的斑马鱼的再生能力。为了验证糖皮质激素的经典抗炎作用是否与阻断再生有关，我们通过反义抑制Pu.1基因来预防截肢后的急性炎症。虽然Pu.1的丢失阻止了炎症反应，但再生不受影响。总之，这些结果表明，外源性糖皮质激素的信号通过急性炎症无关机制损害胚芽形成并限制再生能力。这些研究也证明了利用化学遗传学来确定脊椎动物再生途径的可行性。
Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.